1. Field of Endeavor
The present invention relates to X-ray crystallography and more particularly to automated macromolecular crystallization screening.
2. State of Technology
U.S. Pat. No. 5,597,457 for a system and method for forming synthetic protein crystals to determine the conformational structure by crystallography to George D. Craig, issued Jan. 28, 1997 provides the following background information, “The conformational structure of proteins is a key to understanding their biological functions and to ultimately designing new drug therapies. The conformational structures of proteins are conventionally determined by x-ray diffraction from their crystals. Unfortunately, growing protein crystals of sufficient high quality is very difficult in most cases, and such difficulty is the main limiting factor in the scientific determination and identification of the structures of protein samples. Prior art methods for growing protein crystals from super-saturated solutions are tedious and time-consuming, and less than two percent of the over 100,000 different proteins have been grown as crystals suitable for x-ray diffraction studies.”
International Patent No. WO0109595 A2 for a method and system for creating a crystallization results database to Lansing Stewart et al., published Feb. 8, 2001, provides the following background information, “Macromolecular x-ray crystallography is an essential aspect of modern drug discovery and molecular biology. Using x-ray crystallographic techniques, the three-dimensional structures of biological macromolecules, such as proteins, nucleic acids, and their various complexes, can be determined at practically atomic level resolution. The enormous value of three-dimensional information has led to a growing demand for innovative products in the area of protein crystallization, which is currently the major rate limiting step in x-ray structure determination. One of the first and most important steps of the x-ray crystal structure determination of a target macromolecule is to grow large, well diffracting crystals with the macromolecule. As techniques for collecting and analyzing x-ray diffraction data have become more rapid and automated, crystal growth has become a rate limiting step in the structure determination process.”
U.S. Pat. No. 6,368,402 for a method for growing crystals to George T. DeTitta et al. issued Apr. 9, 2002, provides the following background information, “A number of investigators have attempted to condense their experiences in the crystal growth laboratory into a list of recipes of reagents that have found success as crystallizing agents. The most used of these is the list compiled by Jancarik, J. and Kim, S.-H. (1991), J. Appl. Cryst. 24, 409-411 which is often referred to as the “sparse matrix sampling” screen. The list is a “heavily biased” selection of conditions out of many variables including sampling pH, additives and precipitating agents. The bias is a reflection of personal experience and literature reference towards pH values, additives and agents that have successfully produced crystals in the past. Commercialization of the sparse matrix screen has led to its popularity; easy and simple to use, it is often the first strategy in the crystal growth lab. The agents chosen by Jancarik and Kim are designed to maximize the frequency of precipitation outcomes for a broad variety of proteins. They were chosen because in a large percentage of experiments employing them “something happened.”
U.S. Pat. No. 5,961,934 for a dynamically controlled crystallization method and apparatus and crystals obtained thereby to Leonard Arnowitz and Emanuel Steinberg, issued Oct. 5, 1999, provides the following background information, “The concept of rational drug design involves obtaining the precise three dimensional molecular structure of a specific protein to permit design of drugs that selectively interact with and adjust the function of that protein. Theoretically, if the structure of a protein having a specified function is known, the function of the protein can be adjusted as desired. This permits a number of diseases and symptoms to be controlled. For example, CAPTOPRIL is a well known drug for controlling hypertension that was developed through rational drug design techniques, CAPTOPRIL inhibits generation of the angiotension-converting enzyme thereby preventing the constriction of blood vessels. The potential for controlling disease through drugs developed by rational drug design is tremendous. X-ray crystallography techniques are utilized to obtain a “fingerprint,” i.e. the precise three-dimensional shape, of a protein crystal. However, a critical step to rational drug design is the ability to reliably crystallize a wide variety of proteins. Therefore, a great deal of time and money have been spent crystallizing proteins for analysis.”
International Patent No. WO02/26342 for an automated robotic device for dynamically controlled crystallization of proteins to Leonard Arnowitz et al., published Apr. 4, 2002, provides the following background information, “There is a pressing need for reliable, high yield, high quality crystallization procedures for rational/structural drug design. Existing screening methods including traditional vapor diffusion experiments, automated systems, and commercial screens are inadequate. For example, once a vapor diffusion experiment is set up with a target concentration of the precipitant used, it cannot be modified. This prolongs the optimization process, and makes it nearly impossible to screen effectively a large number of conditions without a large time commitment and large quantities of protein.
Despite its increasing commercial importance, the science underlying crystal growth is incomplete and it is nearly impossible to predict the conditions under which a newly studied protein will crystallize (Ries-Kautt, M., et al., Inferences drawn from Physicochemical Studies of Crystallogenesis and the Precrystalline State: Macromolecular Crystallography. Methods, Eizzyinol. 1997, 276, 23-59; McPherson, A., Crystallization of Biological Macromolecules: Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y., 1999; Ducruix. A., and Giege R., Eds., Crystallization of Nucleic Acids and Proteins: A Practical Approach; ML Press: Oxford, United Kingdom, 1992). The process of protein crystal growth can be dissected into primary nucleation, and nucleation and growth of its chemically and geometrically constituent layers and rows.
Proteins are typically half water by volume, mechanically fragile, and marginally stable. They are prepared in aqueous solutions and crystals of about 0.2 mm size are usually grown for x-ray diffraction measurements that can lead to detailed structural information. Protein crystals are generally cultivated by slowly dehydrating a solution of the pure protein with a large excess of a selected soluble salt or alcohol called the precipitant, and in addition some lower-concentration co-solutes such as buffers and reducing agents that maintain protein stability. Frequently, crystal growth will display a strong sensitivity to these co-solutes due to interactions with the protein surface. In defiance of systematic science, knowing the conditions that gave crystals for one protein does little to predict conditions for even a closely related protein, and a wide assortment of chemical additives has been found useful for various proteins (Jankarik, T., Kim, S. H. Sparse Matrix Sampling: A Screening Method for Crystallization of Proteins. J AppL Cryst. 1991, 24, pp. 409-411). Due to these sensitivities and the instabilities of the proteins themselves, the process of protein crystal growth nearly always involves searching and scanning to optimize the results (Cudney, R., et al., Screening and Optimization Strategies for Macromolecular Crystal Growth. Acta Cryst. 1994, D50, pp. 414-423) For a new protein, the initial search phase involves a broad scan covering wide arrays of potential additives to identify those that promote crystal growth. In the subsequent optimization phase, the scanning is localized around the conditions that have been found to produce crystals.
Efforts to produce crystals, and then to optimize them for diffraction and structure determination, typically take months to years. Crystallization of a biological molecule such as a protein involves the creation of a supersaturated solution of the molecule under conditions that promote minimum solubility and the orderly transition of the molecules from the solution into a crystal lattice. The variables that must be controlled precisely to promote crystal growth include temperature, protein solution concentration, salt solution concentration, pH, and gravitational field, for example (Durbin, S., Felier, G., Ann. Rev. Phys. Chem., 1996, 47, pp. 171-204). These variables are carefully controlled and optimum combinations thereof are determined through experimentation to yield superior crystals.
Removal of water from the protein solution is usually effected by vapor diffusion, by dialysis, or by direct mixing with hypertonic media (Weber, -P. C. Overview of Protein Crystallization Methods: Macromolecular Crystallography. Methods EnrynioL 1997, 276, pp. 13 22). Dialysis methods use a hygroscopic reservoir to remove water. A polysaccharide semipermeable membrane is utilized that blocks passage of the protein but permits water and precipitant to pass through (in opposite directions). A key practical difference is that in vapor diffusion the (usually nonvolatile) precipitant must be premixed with the protein, whereas for dialysis no premixing step is needed since the precipitant can usually traverse the membrane.
Ordinarily the dialysate is not changed (static dialysis) and the precipitant level in the protein chamber increases rapidly at fast, then more slowly. If the precipitant concentration rises too quickly, excess nucleation occurs, while if the trajectory is slower, growth of a few crystals may be favored. If the rise in precipitant concentration occurs too slowly, competing processes like protein aggregation and denaturation may interfere. With direct mixing or batch methods, the protein solution is simply mixed with a precipitant solution. This does not formally remove water, but like the other methods, it raises the concentration of precipitant in the protein compartment and thus decreases protein solubility to promote crystal growth.
A DCCS™ dialysis-based reactor for protein crystal growth, featuring dynamic control of the dialysate is described in commonly-owned U.S. Pat. No. 5,961,934, and in International Publication No. WO 99/4219 1, the contents of which are incorporated herein by reference in their entirety. An embodiment comprises a crystallization chamber that is divided by a semipermeable membrane into two compartments, a reactant chamber and a reagent chamber. The reactant chamber is filled with a reactant solution, e.g., a solution of protein to be crystallized, and the reagent chamber is filled with a reagent solution, e.g., a precipitant solution. The device works by pumping precipitant-rich reservoir solution through the reagent chamber so as to gradually raise the precipitant level experienced by the protein, under computer control. The reagent solution is connected via tubing to a precipitant supply syringe and also to a drain syringe that begins empty.
The existing DCCS™ technology, although an improvement over all prior techniques, is nonetheless time consuming and requires substantial manpower. With the advent of proteomics, and recent advances in cloning methodology, purification techniques, subsequent data collection, and structural determination procedures, a major bottleneck in the structural determination of proteins is the process of protein crystallization. The Protein Structure Initiative has set the goal of crystallizing and determining the structures of 10,000 proteins in the next 10 years. For the Protein Structure Initiative to be a success, novel crystallization procedures are required to permit the structural determination of a large number of proteins in a high throughput mode. There is therefore a strong need for a crystallization system that allows many independent crystallization conditions to be simultaneously screened and dynamically controlled, with minimal repetition, to improve the screening process and enable crystallization of a large number of proteins independently, and quickly, using parallel processing.
With so many important proteins to be crystallized, there is a need for automation of the crystallization experiments. Robotics enables systematic pipetting of solutions and protein into crystal growth chambers on plates, so that a multiplicity of conditions can be examined more quickly and consistently. The use of robotics provides accuracy and frees valuable time for researchers. Another trend is the use of semi-automated techniques to record results. However, these steps do not avoid the bottleneck of the crystallization process itself.
Protein structure research requires not only diffractable protein crystals, but also protein crystals soaked in solutions of heavy atoms, to allow the heavy atoms to bind to the crystallized protein molecules. The resulting heavy atom derivative is useful in obtaining the phase solution needed to determine the structure of the protein. Problems arise because the heavy atom compounds may damage the crystal if present at too high a concentration. In addition, the heavy atom compounds often have low solubility, and so must be used at low concentrations that are barely above the minimum concentration permitting effective binding to the protein. Therefore, as the heavy atom compounds bind the protein and come out of solution, the effective concentration is reduced and further binding is curtailed. Static solutions are typically used to make heavy atom derivatives. It is impossible to predict the optimal concentration of heavy atom to use, and much time and protein are lost in determining solution conditions that are suitable for producing heavy atom derivatives. A dynamic high throughput method of heavy metal binding is needed.
Similar problems arise in efforts to bind ligand and drug molecules to crystallized protein molecules M situ. If the concentration of ligand or drug is too high, it may damage the crystal, however, if it is too low, the ligand or drug will not bind to the protein molecules. As with heavy atoms, the low solubility of many ligand and drug molecules compounds the problem.
Dynamic dialysis devices may help by permitting the concentration of heavy atom compound, ligand, or drug to be gradually increased in a controlled manner. By observing the crystal through a transparent side of the crystallization chamber, damage to the crystal might be detected at an early stage, and the concentration of heavy atom compound, ligand, or drug lowered accordingly. By constantly refreshing the reactant solution, the concentration of heavy atom compound, ligand, or drug can be held constant, even though molecules leave solution upon binding to the protein molecules. However, to test a large number of different combinations of proteins and reactant solutions to produce crystals, heavy atom derivatives, or in situ complexes of crystallized protein and ligand or drug, insuperable difficulties are encountered in attempting to monitor the states of the samples in each reactant chamber, and to adjust the concentration of reagent in response to the observed states of the samples. The more samples that are screened, and the more closely the samples are monitored, the greater the difficulties encountered. There is thus a need for an improved, automated system for dynamically controlled crystallization of proteins by dialysis.”